Intraspecific Variation in Viola suavis in Europe ... - Annals of Botany

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450 A 90 B 81 Estimation of pollen fertility 0·001 changes To test the assumed hybrid origin of the white-flowered morphotype(s), all plants included in morphometric analyses were also examined for pollen viability. Anthers were removed from a single flower or a flower bud per individual and chopped in a drop of aceto-carmine jelly (Radford et al., 1974) on a microscope slide to release pollen grains. One hundred pollen grains per individual were observed. The numbers of unviable (i.e. unstained and usually shrunken) and viable (well-stained and of regular shape) grains were recorded. Pollen quality was expressed as the percentage of viable grains. RESULTS Karyological analyses Data on chromosome number and DNA ploidy level are presented in Appendix. All populations of V. alba, V. collina, V. hirta and V. odorata were found to be tetraploid with 2n ¼ 20, while all populations of V. suavis (both morphotypes) were octoploid with 2n ¼ 40. These data are consistent with previous reports (cf. Mered’a et al., 2006; Hodálová et al., 2008). Molecular analyses ITS sequences. The ITS matrix excluding the outgroup consisted of 38 sequences and 612 aligned positions; 37 sites were variable; 34 of them were parsimony informative. A few 1-bp or 2-bp indels were identified, which were not coded separately but treated as missing data. Only a few sequences contained apparent intra-individual singlesite polymorphisms (overlapping peaks in electropherograms; up to two positions per sequence). The parsimony analysis (figure not shown) resulted in a strict consensus tree (2288 most parsimonious trees; L ¼ 50 steps, Mered’a et al. — Intraspecific Variation in Viola suavis 71 100 100 AMB 149a AMB 149b 95 AMB 151a 59 AMB 151c AMB 151b AMB 149c 65 62 AMB 21a AMB 21c AMB 21b 76 ambigua CI ¼ 0.92, RI ¼ 0.98), which displayed a large polytomy at the base. Split decomposition analysis revealed that this is due to data conflict; the splits graph (Fig. 2) indicates that patterns of nucleotide substitutions are incompatible, and the relationships among the species are uncertain. In addition, no sequence divergence was found between V. collina and V. odorata. The monophyletic character of V. suavis, including both morphotypes, is evident. AFLP fingerprinting. In the 178 individuals studied, 288 unambiguous AFLP fragments were scored; 32 fragments (11 %) were monomorphic across the dataset. Control replicates proved that the AFLP data were highly reliable (repeatability 98.6 %). Altogether, 77 different AFLP multilocus phenotypes were detected, i.e. several individuals had identical profiles. Table 2 shows the number of AFLP phenotypes and the average number of fragments per individual that were resolved in the taxa studied. In the two octoploid species, V. ambigua and V. suavis, more fragments per individual were generated than in the tetraploids, indicating a correlation with the ploidy level. The neighbor-joining tree (Fig. 3) and PCoA (figure not shown) based on the ‘all species matrix’ resolved six distinct and well-differentiated groups, corresponding to the six species studied. This result was also supported by the number of species-specific fragments (Table 2). Clustering in the neighbor-joining analysis suggests closer affinities between certain species but with moderate to low bootstrap support. With respect to variation patterns within V. suavis (both morphotypes), neighbor-joining clustering, PCoA and STRUCTURE analyses (the latter two based on the ‘V. suavis matrix’) revealed a pronounced intraspecific genetic structure. In PCoA (Fig. 4), the accessions from Spain were clearly separated from those from C & SE Europe along the first axis, which extracted almost 32 % of the total variation. This separation was seen also in the neighbor-joining tree (Fig. 3). Consequently, 100 HIR 198a HIR 198b HIR 198c 93 HIR 145a 71 HIR 145b HIR 145c hirta 92 HIR 142a 75 HIR 142b HIR 142c COL 130a COL 130bCOL 62 30a COL 130c 100 78 COL 127a COL 127b COL 30c COL 127c COL 30b ODO 178a 90 ODO 178b ODO 178c 100 79 ODO 215a 89 ODO 215b ODO 215c odorata ODO 146a ODO 146b ODO 146c F IG. 3. (A,B) Midpoint rooted neighbor-joining tree of AFLP data (288 fragments) of 178 Viola individuals using Nei and Li distance. Numbers above branches indicate bootstrap support above 50 %. Accession labels include taxon abbreviation (ALB, V. alba; AMB, V. ambigua; ODO, V. odorata; COL, V. collina; HIR, V. hirta; SUB, V. suavis, blue-flowered morphotype; SUW, V. suavis, white-flowered morphotype) and population numbers (see Appendix). collina Downloaded from http://aob.oxfordjournals.org/ by guest on March 20, 2013
90 A 0·001 changes white-flowered accessions appear clearly biphyletic. Iberian ones (five populations in total) all shared a single AFLP phenotype, which appeared close to those resolved in Iberian blue-flowered populations. The white-flowered populations from C & SE Europe formed a separate Mered’a et al. — Intraspecific Variation in Viola suavis 451 1 = 2 = 3 = 4 = 100 suavis – white C & SE Europe suavis – blue C & SE Europe suavis – white Spain suavis – blue Spain F IG.3. Continued ALB 190a ALB 188a SUW 106a SUW 106b SUW 106d SUW 106e SUW 143a SUW 143b SUW 143c SUW 147b SUW 147c SUW 201a SUW 201b SUW 201c SUW 201d SUW 201e SUW 205a SUW 205e SUW 207a SUW 207c SUW 207d SUW 207e SUW 208a SUW 208b SUW 208d SUW 208e SUW 22a SUW 22b SUW 22c SUW 22d SUW 22e SUW 7a SUW 7b SUW 7c SUW 7d SUW 7e SUW 207b SUW 208c SUW 106c SUW 205c SUW 205d SUW 147a SUW 147d SUW 205b SUB 206a SUB 206b SUB 206c SUB 212a SUB 212b SUB 212c SUB 212d SUB 212e SUB 211a SUB 211b SUB 211d SUB 206d SUB 206e SUB 209a SUB 209b SUB 209c SUB 209d SUB 122a SUB 122b SUB 122c SUB 122d SUB 129a SUB 129b SUB 129c SUB 129d SUB 129e SUB 181a 97 SUB 181d SUB 181e 69 SUB 181b SUB 181c SUB 193a 100 SUB 193b SUB 193c SUB 193d 58 SUB 211c SUB 211e SUB 216a 91 93 SUB 148a SUW 163a SUW 168a SUW 168b SUW 168c SUW 168d SUW 168e 100 100 SUW 170a SUW 170b 100 SUW 170c SUW 170d SUW 170e SUW 171a SUW 171b SUW 171c SUW 171d SUW 171e SUW 175a SUW 175b SUW 175c SUW 175d SUW 175e SUB 165a 100 100 SUB 169a 65 ALB 190b ALB 188c ALB 179a ALB 188b ALB 179b ALB 190c ALB 179c alba 1 SU 25a SU 25e 61 5366 75 61 85 74 67 SUB 206f SU 25c SU 25d 95 97 SUB 216b SUB 216c SUB 216d SUB 216e SU 25b SUW 163b SUB 148b SUB 148c SUB 148d SUB 148e 68 SUB 4a SUB 4d SUB 4e SUB 4b 67 SUB 4c 100 50 SUB 165b SUB 165c SUB 165d SUB 165e SUB 165f SUB 169b 100 SUB 174a SUB 174b SUB 174c 3 4 subcluster among the blue-flowered accessions from the same area (Figs 3 and 4). Results of the Bayesian clustering (STRUCTURE) of V. suavis are in good agreement with the phenetic analyses. The assignment of individuals into clusters across replicate 2 Downloaded from http://aob.oxfordjournals.org/ by guest on March 20, 2013
Page 1 and 2: Annals of Botany 102: 443-462, 2008
Page 3 and 4: the variation in the population is
Page 5 and 6: and an incubation at 4 8C (Mastercy
Page 7: V. suavis - blue (211, 212) V. suav
Page 11 and 12: TABLE 3. Analysis of molecular vari
Page 13 and 14: due to isolation by distance. Sampl
Page 15 and 16: elationship between tetraploid (2n
Page 17 and 18: Vos P, Hogers R, Bleeker M, Reijans
Page 19 and 20: APPENDIX. Continued Taxon, pop. no.

Intraspecific Variation in Viola suavis in Europe ... - Annals of Botany

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